The rational design and construction of a cuboidal iron–sulfur protein

Coldren, Christopher D.; Hellinga, Homme W.; Caradonna, John P.

doi:10.1073/pnas.94.13.6635

Cited by 98 publications

(73 citation statements)

References 38 publications

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“…Examples of such peptides include Cu(II)-binding motifs related to Gly-Gly-His, [93][94][95][96] as well as small peptides that assemble into Fe 4 S 4 clusters. 97,98 However, complete control of a cofactor's environment might be best effected through the de novo design [99][100][101][102] of proteins whose active sites are defined by the favorable free energy of folding of the polypeptide chain. There has also been much progress made in the de novo design of proteins that bind metalloporphyrins [92][93][94][95][103][104][105] and Zn-(II).…”

Section: From Structure To Function: Design Of Metalloproteinsmentioning

confidence: 99%

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

et al. 2000

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De novo protein design has proven to be a powerful tool for understanding protein folding, structure, and function. In this Account, we highlight aspects of our research on the design of dimeric, four-helix bundles. Dimeric, four-helix bundles are found throughout nature, and the history of their design in our laboratory illustrates our hierarchic approach to protein design. This approach has been successfully applied to create a completely native-like protein. Structural and mutational analysis allowed us to explore the determinants of native protein structure. These determinants were then applied to the design of a dinuclear metal-binding protein that can now serve as a model for this important class of proteins.Protein folding is an important and multifaceted scientific problem. 1-8 One facet of this problem involves the prediction of three-dimensional structure from amino acid sequence, an endeavor the importance of which has been highlighted by the recent release of gene sequences of many whole organisms. As the size of the protein structural database expands, it will be increasingly possible to assign amino acid sequences to a given structural family on the basis of the homology of their sequences with proteins of known structure. Another facet of the folding problem is the delineation of the kinetic mechanism by which an unfolded protein chain undergoes the transition from a random coil with an astronomical number of configurations to a uniquely folded structure. A final facet of the protein folding problem is to understand, at the atomic level, the detailed physicochemical features that kinetically and thermodynamically direct the formation of an unique, cooperatively folded, native structure. This latter endeavor is particularly important as it lays the groundwork for the design of novel inhibitors of natural proteins as well as the construction of novel proteins and related biopolymers not found in nature.De novo protein design is an approach to the protein folding problem that critically tests our understanding of all these facets of this problem. 9,10 This method involves the design of a sequence that is intended to fold into a predetermined three-dimensional structure without the sequence being patterned after any natural protein. Through an iterative process of design and rigorous characterization, the principles governing protein folding and function can be evaluated. Thus, "failures" highlight gaps in our understanding, whereas "successes" confirm the principles used in the design and often provide simple model systems to further

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Section: From Structure To Function: Design Of Metalloproteinsmentioning

confidence: 99%

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

et al. 2000

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“…A variety of metal sites have been designed into thioredoxin. For example, one mimics the active site of iron-superoxide dismutase (Pinto et al, 1997), while another introduces a [Fe4S4] cuboidal cluster into the hydrophobic core (Coldren et al, 1997). Several other designs have incorporated [Fe4S4] cuboidal clusters into the loops of four-helix bundle proteins (Gibney et al, 1996;Scott & Biggins, 1997).…”

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The de novo design of a rubredoxin‐like fe site

Farinas

Regan

1998

Protein Science

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A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B 1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [ F e ( S -c~s )~] ",-'. The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(I1) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(I1)-bound forms of the variant are folded correctly and are stable. The Fe(II1) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(II1) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.Keywords: de novo design; metalloprotein; protein engineering; rubredoxin Protein engineering and de novo design approaches have begun to afford a more detailed understanding of the balance of the forces that determine protein structure and function (Regan & DeGrado, 1988;Regan, 1993;Bryson et al., 1995;Regan, 1995;Dalal et al., 1997;Smith & Regan, 1997;Hellinga, 1998). To date, there are just a few examples of designed proteins that adopt a specified fold, and number which display novel activities is still smaller (Dill, 1990; Schafmeister et al., 1993;Jackson et al., 1994;Quinn et al., 1994;Yan & Erickson, 1994;Baltzer et al., 1996;Dahiyat & Mayo, 1997;Hill & DeGrado, 1998;QuCnCneur et al., 1998). A particular focus of functional design efforts has been the engineering of novel metal-binding sites. Metal-binding sites represent an attractive target because they play a variety of functions from stabilization of protein structure to catalytic roles such as the activation of water for nucleophilic attack, electron transfer, and redox activity. More recently, several groups have focused strongly on the design of metal-binding sites with particular activities in mind. For example, two metal-binding histidine residues have been engineered into rat trypsin to "switch" the catalytic activity on or off by disrupting the catalytic triad. The protein can be turned off by Cu(I1) chelation in the designed site and reactivated upon removal of Cu(I1) with EDTA (Halfon & Craik, 1996). In another design, a metal-binding site, based on the metal center of carbonic

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“…Automated methods have been developed recently for engineering such ion-binding sites (15, 16), and it has been possible to build a number of structural as well as redox-active metal ion-binding sites within several different proteins (17)(18)(19)(20). Another successful approach has been to design small flexible peptides that are able to fold around metal sites such as Cu(II)-binding motifs (21-24) and small peptides that assemble into Fe 4 S 4 clusters (25,26).However, complete control of a cofactor's environment might be best effected through the de novo design (27, 28) of proteins whose active sites are defined by the favorable free energy of folding of the polypeptide chain. Unfortunately, initial attempts to design proteins led to structures that formed molten globulelike states with dynamic behavior relative to natural proteins.…”

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confidence: 99%

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Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

Lombardi

Summa

Geremia

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

233

279

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De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 Å rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the GluXxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 Å. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation. P roteins use a limited repertoire of metal ion cofactors to help catalyze a multitude of reactions. For example, diiron sites (1-4) mediate reversible oxygen binding in hemerythrins, whereas they function as hydrolytic centers in phosphatases. Structurally similar diiron sites also mediate a number of oxygendependent oxidative processes. Ferritins serve as ferroxidases, while other diiron proteins catalyze hydroxylation, epoxidation, and desaturation reactions. Further, a diiron site in Escherichia coli ribonucleotide reductase is responsible for the formation of a Tyr radical. How do the structures of these proteins tune the chemical properties of a common diiron center to obtain such a diversity of highly specific catalysts? This question is being addressed through the study of the natural proteins as well as the study of small-molecule diiron complexes (1-4). Although impressive progress has been made on both fronts, these approaches have inherent limitations. The study of large proteins is hampered by their extreme complexity, and it is difficult to synthesize small-molecule models capable of simultaneously binding diiron, oxygen, and various substrates. Recently, we and others have sought a molecular middle ground between these two extremes through the design of small proteins and peptid...

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The rational design and construction of a cuboidal iron–sulfur protein

Cited by 98 publications

References 38 publications

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

The de novo design of a rubredoxin‐like fe site

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

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